The soils from different fertilizers application treatments in the long-term experiment on red soil upland were analyzed. The results showed that the effect of the treatments with different fertilizers application on soil characteristics in red soil upland was very significant. The soil labile organic matter decreased 39% after applying chemical fertilizers only for 13 years. It increased 16.9% for the NPKS treatment, and 11% for M. The amount of soil microbes and the activity of soil enzyme in the treatments with long-term application of straw and organic manure were higher significantly than that in the treatments with long-term application of chemical fertilizers, and it was the lowest in the treatment with long-term application of chemical N fertilizer only. The soil was severely acidified and the yield of corn was obviously decreased after long-term applying chemical fertilizers on red soil upland. The results of average crop biomass yields from 13 years showed that the yield of N, NPK, NPKS, M and NPKM treatment increased 24.9%, 267%, 319%, 267% and 367%, respectively, compared with control treatment. Applying straw and organic manure could prevent soil acidification, and enhance the availability of soil nutrients and utilization efficiency of fertilizers.